ThisproposalaimstotestthehypothesisthattypeIIafferentsserveascochlearnociceptors.Takingcuesfromthehumancomplaintofhyperacusisafterhearingloss,wewillexaminethestructureandfunctionoftypeIIafferentsinnormalandpost-traumacochleas.Theworkinghypothesisisthatpainfulhyperacusis,noxacusis,includeshyperactivityoftypeIIafferents,byanalogytohyperalgesiaofsomatic nociceptive C-fibers.ThuswewillexaminetypeIIstructureandfunctioninnormalandpost-trauma cochleasofratsandmice.Inparallelwewillinvestigatethepropertiesofsurvivingouterhaircellsinposttraumacochleas.Ourmethodsinclude:exvivoelectrophysiology,lightandelectronmicroscopy,utilizationofoptogeneticandchemogenetictools,andvalidationandquantificationofmousemodelsinwhichtypeIIspecificbio-markersareexpressed.Anecessaryfirststepistoextendourexvivoexperimentalapproachtooldercochleassothatchangeswroughtbyacoustictraumacanbecomparedtothenormalcondition.Wewillcomparedamagingsound,ototoxicantibioticsandgeneticallyencodedbiotoxinstoproduceexperimentallytractableeffectsontissueforexvivoexperiments.ThepropertiesandsynapticconnectionsoftypeII afferentsandouterhaircellswillbeexaminedintheexcisedcochleartissueoftheseanimals.Wewill continuetoexploretypeIIspecificgeneticmousemodels.Genetically-encodedreporterproteins, voltage-andcalcium-sensitiveindicators,biotoxins,andopto-andchemo-geneticmodulatorshave becomehighlyinformativetoolsinneurobiologygenerallyandfortheinnerearspecifically.Ourongoingworkhascharacterizedonemouseline,tyrosinehydroxylasepromoterdrivenCre-recombinaseexpression.ThreeothercandidatetypeIIspecificCrelineswillbevalidatedandquantified.Withsuchtransgenicmodelsitbecomespossibletostudyinnervationpatternsbyexpressionoffluorescentreporterproteins,andtoactivate,eliminate,ormodulatetypeIIactivityforanatomicalandphysiologicalstudies. Cre-dependentexpressionofgenetically-modifiedG-protein coupledreceptors (DREADDS) willprovide mice in which type II activity can be increased or decreased by injection of anovelsyntheticligand,dependingonthespecificconstruct.Varyingcombinationsofsystemicandroundwindowdrugdeliverywillbeemployedtoincreasethespecificityofexperimentalmanipulations.Theover-archinggoalofthisprogramofexperimentsistocompletethedescriptionoftypeII afferents,astill-unresolvedcomponentofcochlearinnervation. Theworkinghypothesisisthatthese serveascochlearnociceptors. Ifcorrectthesearealikelyneurobiologicalsubstratefornoxacusis (painfulhyperacusis). BydefiningthebasiccellularandmolecularmechanismsoftypeIIfunctionand plasticity,futuretherapeutictargetscanbeidentifiedtoameliorateorpreventnoxacusis.